Name: indirect immunofluorescence on frozen sections of mouse mammary gland
Uploaded: 2015-12-01T14:00:00
Description: Watch this Scientific Journal Video about Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland at JoVE.com

Forfatterne er takknemlige til INRA MIMA2 bildebehandling kjernefasiliteten (INRA, UMR1198, Jouy-en-Josas) og til de ansatte på IERP enheten (UE 0907, INRA, Jouy-en-Josas) for dyr omsorg og fasiliteter. Vi vil også gjerne takke IH Mather, MC Neville og S. Tooze for å gi oss svært nyttig antilegemer.

CD1 mus ble avlet på INRA (UE0907 IERP, Jouy-en-Josas, Frankrike). Alle etiske aspekter av dyr vare oppfylt de relevante retningslinjer og konsesjonskrav fastsatt av franske Landbruksdepartementet. Prosedyrene som benyttes ble godkjent av den lokale etikkutvalg (avtalen 12/097 fra Comethea Jouy-en-Josas / AgroParisTech). 1. melkekjertler Prøvepreparering <ol><li> Mus melkekjertler disseksjon <ol><li> Avlive mus på dag 10 av amming ved halshugging og pin dyret ned med buken opp....

IHC er en forholdsvis enkel og grei eksperimentelle metoden for å lokalisere antigenet i vevssnitt, som avhenger primært av spesifikke epitop-antistoff-interaksjoner. Selv om et stort antall protokoller brukes for å lokalisere et protein ved IIF, er kjernen i disse prosedyrene nesten alltid den samme. Det er imidlertid noen kritiske aspekter som sterkt kan påvirke resultatet, og må derfor være optimalisert for hvert enkelt IHC studium. Den mest utfordrende aspekt ved denne tilnærmingen er å bestemme de beste eks...

Melkekjertlene er et atypisk pattedyr eksokrin organ hvis viktigste funksjon er å produsere melk til å mate nyfødte. Utviklingen av brystvev forekommer hovedsakelig etter fødselen, og er karakterisert ved en unik fremgangsmåte hvor epitelet invaderer den omkringliggende stroma. Dette vevet gjennomgår mange endringer (vekst, differensiering og regresjon), spesielt i løpet av voksenlivet, samtidig med variasjoner i reproduktiv status (figur 1). I tillegg til den totale morfologi av vev, andelene av forskjellige celletyper, så vel som deres arrangement i melkekjertelen dramatisk endre seg under utvikling 1-5. I løpet av embryonisk liv, utleder brystepitel fra brystmelkeledninger, som er definert ved en svak fortykning og lagdeling av ektoderm, mellom for- og baklemmene på hver side av midtlinjen rundt embryodag 10,5 (E10.5) (figur 1A ).På E11.5, melk linjen bryter opp i individuelle placodes, som er symmetrisk plassert langs melke melk linjen på reproduserbare steder, og det omkringliggende mesenchyme begynner å kondensere. De placodes begynne å synke dypere inn i dermis og melke mesenchyme organiserer i konsentriske lag rundt melke bud (E12.5-E14.5). Per E15.5, melke epitel, begynner å spre seg og forlenge å danne primære spire som presser gjennom melke mesenchyme mot fettpute. Den primære spire utvikler en hul lumen med en åpning på huden, som er merket ved dannelsen av nippelen kappen. På E18.5 har elongating duct vokst til fettpute og har forgrenet inn i en liten arborized ductal systemet omfattet i fettpute. Utviklingen er i hovedsak arrestert og rudimentær melkekjertlene forblir morphogenetically hvilende inntil puberteten. I den mannlige foster, aktivering av androgen-reseptorer fører til degenerering av knopper, som forsvinnerved E15.5. Per E18, mammary utvikling opphører før puberteten 6-9. Ved fødselen, havner melkekjertlene en rudimentær duktal system som forlenges og grener sakte (isometrisk vekst). Ved utbruddet av pubertet, sfæriske strukturer som ligger på tuppen av kanalene kalles terminal endeknopper (TEBs), dannes av et ytre lag av cap celler og et flerlags indre kjerne av celler (celler i kroppen). Disse strukturene er svært proliferativ og infiltrere omliggende stromal vev som svar på hormonelle signaler. Spredning innenfor TEBs resultater i duktal forlengelse, kombinert med forgrening morphogenesis. Denne prosessen fører til at det etableres en grunnleggende epitelial arborized nettverk som kommer fra nippelen (figur 1B, pubertet). På ~ 10-12 uker etter fødselen, når Epitel har invadert hele fett puten, stopper og sin ekspansjon i TEBs forsvinne. Ductal utvikling gjennomgår deretter dynamiske endringer, dvs. successive spredning og regresjon av epitelceller i henhold til østrogensyklus 10 (figur 1B, voksen). Fra begynnelsen av svangerskapet, gjennomgår brystvev viktig vekst og morfologiske endringer for å forberede laktasjon. Den melkekjertelepitelet omfattende spre og differensiere, fører til et sterkt forgrenet tubulointerstitiell alveolar nettverk. Samtidig, mammary epitelceller (mecs) blir polarisert og i stand til å syntetisere og skiller melkeprodukter. Mecs organisere inn i mange alveolære strukturer (acini) som er omgitt av kontraktile korgcellene og innlemmet i en stroma består av bindevev og fettvev, blodkar og nerveterminaler (figur 1B, graviditet). Videre er det basale siden av mecs i nær kontakt med basalmembranen (ekstracellulær matriks), og vekselvirkning mellom disse to enhetene tett regulere både morfogenese og sekretorisk funksjon av mammary epitel 11-13. Alle disse prosessene er avhengige av virkningen av ulike miljø signaler, hvorav de viktigste er hormones14, parakrine faktorer og ekstracellulære matrise. For eksempel induserer progesteron omfattende sideforgrening 15 og alveologenesis som i kombinasjon med prolaktin (PRL) 16,17, fremmer og opprettholder differensiering av alveolene. I tillegg til steroider og PRL18, cytokiner og signalveier knyttet til utvikling (Wnt og Notch signalveier) er også involvert i bryst avstamning engasjement og utvikling 19-21. Ved slutten av svangerskapet, luminal mecs begynne å produsere et proteinrikt melk kalt kolostrum i lumen av alveolene. I tillegg fungerer progesteron på epiteliale permeabilitet og siden de tett veikryss er fortsatt åpne, er råmelk også funnet i mors blod stream. Etter fødsel, den Mammary epitelet opptar nesten hele brystkjertelen volum og er høyt organisert (figur 2, brystepitel). Melkeproduserende enheter, nemlig alveoler (figur 2, tannhulen), er dannet av et monolag av polariserte mammary epiteliale sekretoriske celler (MESCs), med sin apikale plasmamembranen som avgrenser hulrommet. Alveolene ordne seg inn lobules som er gruppert i fliker som er koblet til kanaler som drenerer melk til utsiden miljø (figur 2, lapp). Amming oppstår, altså., MESCs begynner å utskille rikelige mengder melk, først og fremst utløst av fallet i placenta hormoner (hovedsakelig progesteron) (figur 1B, amming). Melk-proteingenene er aktivert i en tidsmessig definert tidsforløp som strekker seg fra graviditet til amming 9,22,23, hovedsakelig som svar på hypofysen PRL frigjøres ved suging. Samtidig, kontakter mellom MESCs og ekstracellulære matrise både stimulerer melkeprotein synthesis gjennom signaler som formidles via interaksjoner mellom cellulære inte og laminin 24,25, og undertrykke apoptose i MESCs 26,27. Disse signalveier resultere i aktivering av melkeprotein genet promotorer 28 gjennom aktivering av spesifikke transkripsjonsfaktorer 29. Celle-celle-kontakter er også viktig for noen aspekter av differensiering, inkludert etablering av apikal polaritet og vektor sekresjon av melkeprodukter. Tett veikryss raskt tett etter begynnelsen av laktasjonen og MESCs fint organisere opptaket av molekyler fra blodet, så vel som syntesen, transport og sekresjon av melkekomponenter, i respons til de ernæringsmessige behov for nyfødte. På tidspunktet for suging, oppstår sammentrekning av korg celler som omgir alveolene i respons til oxytocin og fører til melke utstøting gjennom kanalene og inn i nippelen. Melk er en sammensatt væske som inneholder proteiner (for det mestekaseiner), sukker (hovedsakelig laktose), lipider og mineraler, samt bioaktive molekyler slik som immunoglobuliner A (IgA), vekstfaktorer og hormoner. Kaseiner blir syntetisert, samlet i supramolekylære strukturer, nemlig casein miceller, transporteres langs den sekretoriske vei, og deretter frigjøres ved exocytose, dvs. blanding av kasein inneholdende sekretoriske vesikler (SVs) med den apikale plasmamembranen til Mesc (figur 2). Intracellulær trafikk er avhengig av materielle utveksling mellom membran avdelinger og innebærer Løselig N-etylmaleimid-Sensitive Fusion (NSF) Vedlegg Protein (SNAP) Receptor (SNARE) 30,31. Snaren proteiner familien er inndelt i vesikulær snarer (v-snarer), til stede i vesikkelmembranen, og målet snarer (t-snarer), lokalisert på målet membraner. Ved som gjennom sine kveil-spiral-domener, V- og T-snarene sammen for å danne en svært stabil fire-heliksbunt kompleks, referert til som the SNARE kompleks. Dette komplekset fremmer fusjon av to motstående lipidbilag ved gradvis å bringe dem i tett nærhet 30,32. Etterpå er snare komplekser dissosiert av NSF adenosin triphosphatase og dets adapter protein SNAP og snare proteiner blir resirkulert tilbake til sitt segment til opprinnelses 33. Interessant, hver SNARE protein hovedsakelig ligger i forskjellige cellulære avdelinger og SNARE sammenkobling kan bidra til spesifisitet av intracellulære fusjons hendelser 34. Tidligere studier antyder at minst synaptosomale tilknyttet protein 23 (SNAP23) og Vesikkel-Associated Membrane Protein 8 (VAMP8), og syntaxins (STX) -7 og -12 spille en rolle i kasein exocytose 35,36. Disse proteinene har også blitt funnet i forbindelse med lipid fraksjon av melk, dvs. melk fettkulene (MFGs) 37. Den nåværende rådende modell postulerer at cytoplasmatiske lipiddråper (CLDs) er dannet ved akkumulering av nøytralt lipids (hovedsakelig triacylglyceroler og sterolestere) og kolesterol avledet fra mors kosthold mellom de to flygeblader i det endoplasmatiske retikulum (ER) membran 38-41. Store CLDs er dannet, i det minste delvis, ved fusjon av mindre CLDs under transport til den apikale siden av MESCs hvor de blir utgitt som MFGs (1-10 mikrometer i diameter) ved knoppskyting, blir omviklet med Mesc apikale plasmamembranen 40-42. Amming opphører etter pups er avvent og MESCs gradvis dør ved apoptose, som fører til regresjon av brystvev tilbake til en pubertets tilstand (figur 1B, involusjon). Immunfluorescens (IF) er et felles analyselaboratorium metoden som brukes i nesten alle aspekter av biologi, både i forskning og i klinisk diagnostikk. IF teknikker kan bli utført på vevssnitt (immunhistokjemi, IHC), eller celle (immunocytokjemi, ICC) prøver. Denne kraftige tilnærming er avhengig av bruk av fluorescent-merkede antistoffer som spesifikt binder (direkte eller indirekte) til antigenet av interesse, og således tillate visualisering av dens vevsfordeling gjennom fluorescens mikroskopi. Fluorescenssignaler for det meste avhenge av kvaliteten og konsentrasjonen av antistoffene og korrekt håndtering av prøven. En enkel indirekte immunfluorescens (IIF) protokoll er presentert for å detektere melkeprodukter (kaseiner og MFGs) og proteiner som er involvert i melkeprodukt sekresjon (butyrophilin (BTN1), snare proteiner) på frosne snitt av mus brystvev (figur 3). Mens denne protokollen gir en komplett IHC oversikt, alt fra vev samling til bilde post-behandling, kritisk og valg trinn samt noen tekniske anbefalinger er også presentert og diskutert.

<table border="0" cellpadding="0" cellspacing="0" style="width:756px;" width="755">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Dissection</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:236px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Pins</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ethanol</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Scissors</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel and adapted blades</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ice</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Towel paper</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Tissue sample preparation</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Phosphate Buffered Saline (pH7.4)</td>
			<td style="width:155px;">Sigma</td>
			<td>P-3813</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Paraformaldehyde (PFA, 32% EM grade, 100 ml)</td>
			<td style="width:155px;">Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>personnal protection equipment required WARNING: this product will expose you to Formaldehyde Gas, a chemical known to cause cancer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT compound/Tissue Tek</td>
			<td style="width:155px;">Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose (D-saccharose)</td>
			<td style="width:155px;">VWR</td>
			<td>27480.294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plastic molds</td>
			<td style="width:155px;">Dominique Dutscher</td>
			<td>39910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Liquid nitrogen</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Cryostat/sample support</td>
			<td style="width:155px;">Leica&#160;</td>
			<td>CM3050S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Razor blades (SEC35)</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td>152200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Slide box</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Glass slides Superfrost/Superfrost Ultra Plus</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td>10143560W90/1014356190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Brushes</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">IHC</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Super Pap Pen</td>
			<td style="width:155px;">Sigma</td>
			<td>Z377821-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Permanent marker (black)</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:239px;">50 mM NH4Cl in PBS</td>
			<td style="width:155px;">Sigma</td>
			<td>A-0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">0.1 M glycine in PBS</td>
			<td style="width:155px;">VWR</td>
			<td>24403.367</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Antigen Retrieval solution: Tris 100 mM 5% urea pH9.6</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Heater (up to 100&#176;C)</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:155px;">Sigma</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Vectashield (anti-fading mounting medium) without DAPI/with DAPI</td>
			<td style="width:155px;">Vector Laboratories</td>
			<td>H-1000/H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Glass coverslips 22x50mm (microscopy grade)</td>
			<td style="width:155px;">VWR</td>
			<td>CORN2980-225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nail polish</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Primary antibodies</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:239px;">Rabbit anti-mouse caseins (#7781; 1:50 dilution)</td>
			<td style="width:155px;">generously gifted by M.C. Neville (University of Colorado Health Sciences 
			Center, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Mouse anti-cytokeratin 8 (CK8, clone 1E8, 1:50 dilution)</td>
			<td style="width:155px;">Biolegend (Covance)</td>
			<td>MMS-162P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Mouse anti-cytokeratin 14 (CK14, cloneLL002, 1:50 dilution)</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td>MS-115-P0/P1</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:239px;">Rabbit anti-butyrophilin (1:300 dilution)</td>
			<td style="width:155px;">generously gifted by I.H. Mather (Department of Animal and Avian Sciences University of Maryland College Park, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:239px;">Rabbit anti-Stx6 (1:50 dilution)</td>
			<td style="width:155px;">generously gifted S. Tooze 
			(Cancer Research UK, London Research Institute, London, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Rabbit anti-VAMP4 (1:50 dilution)</td>
			<td style="width:155px;">Abcam</td>
			<td>ab3348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Secondary antibodies</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;">Rhodamine-conjugated goat anti-rabbit IgG (H + L) (1:300 dilution)</td>
			<td style="width:155px;">Jackson ImmunoResearch Laboratories</td>
			<td>111-025-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Counterstains</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Bodipy 493/503</td>
			<td style="width:155px;">Life Technologies (Molecular Probes)</td>
			<td>D-3922</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">DAPI (4-6-diamidino-2-phenylindole)</td>
			<td style="width:155px;">Life Technologies (Molecular Probes)</td>
			<td>D-1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Observation/Image capture</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:239px;">conventional fluorescence microscope</td>
			<td style="width:155px;">Leica Leitz DMRB 
			microscope</td>
			<td></td>
			<td style="width:236px;">Standard filters for FITC, Rhodamine 
			and DAPI emissions,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#215;63 oil-immersion objective (NA 1.3), DP50 imaging camera (Olympus), Cell&#710;F software (Olympus)</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:239px;">Laser Scanning Microscope (confocal microscopy)</td>
			<td style="width:155px;">Zeiss LSM 
			510 microscope</td>
			<td></td>
			<td style="width:236px;">Plan-Apochromat &#215;63 oil-immersion objective (NA 1.4),&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; CLSM 510 software,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Confocal facilities, MIMA2 Platform, INRA Jouy-en-Josas, France, http://mima2.jouy.inra.fr/mima2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Image treatment</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ 1.49k software</td>
			<td style="width:155px;"></td>
			<td></td>
			<td>Free software</td>
		</tr>
	</tbody>
</table>

indirect immunofluorescence on frozen sections of mouse mammary gland

Indirekte immunfluorescens blir brukt til å detektere og lokalisere proteiner av interesse i et vev. Protokollen som presenteres her beskriver en komplett og enkel metode for immun påvisning av proteiner, musemelkegivende brystkjertel blir tatt som et eksempel. En protokoll for utarbeidelsen av vevsprøver, spesielt om disseksjon av mus melkekjertlene, vev fiksering og frosset vev seksjonering, er detaljerte. En standard protokoll for å utføre indirekte immunfluorescens, inkludert en valgfri antigen gjenfinning trinn blir også presentert. Observasjon av de merkede vevssnittene samt bilde oppkjøp og etterbehandling er også oppgitt. Denne prosedyren gir full oversikt, fra samlingen av animalsk vev på det cellulære lokalisering av et protein. Selv om denne generelle metode kan anvendes på andre vevsprøver, bør det være tilpasset hvert vev / primært antistoff par studert.

Melkekjertlene er en subkutan kjertel som ligger langs ventral strukturen i både brystkassen og magen hos gnagere. Plasseringen av de fem par kjertler fra mus under drektighetsperioden, er vist i figur 4. Morfologien av brystkjertelen endres dramatisk i løpet av sin utvikling, noe som reflekterer funksjonelle modifikasjoner som kreves for å forberede for fullstendig laktasjon (figur 1B). I jomfruelige eller førstegangsfødende dyr, består i melkekjertelen av et sparsom...

Watch this Scientific Journal Video about Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland at JoVE.com

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Den indirekte immunfluorescens protokoll beskrevet i denne artikkelen, tillater påvisning og lokalisering av proteiner i musebrystkjertelen. En komplett fremgangsmåte er gitt for å fremstille melkekjertlene prøver, for å utføre immunhistokjemi, å avbilde vevssnittene ved fluorescens mikroskopi, og å rekonstruere bilder.

Indirekte Immunfluorescens på Frosne Deler av Mouse melkekjertler

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

indirect-immunofluorescence-on-frozen-sections-of-mouse-mammary-gland

Research

JoVE Journal

Biology

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Enkelt Cell transcriptional Profilering av Adult Mouse cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Intraductal Injection for Lokalisert Drug Deli til musemelkekjertlene

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

Effekt av Mann Accessory Gland Produkter på egglegging i gastropod Bløtdyr

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

Isolering av intakt, hele musen brystkjertlene for analyse av ekstracellulær Matrix uttrykk og kjertel morfologi

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...